Homing endonucleases (also termed meganucleases) are highly specific enzymes that are under intense study for the purpose of targeted genome engineering and gene therapy. We have previously determined the structures of representatives from many known homing endonuclease families, characterized their mechanisms of DNA recognition and catalysis, and created variants that cleave noncognate DNA targets. We will now pursue two specific aims that build upon these results and that pursue new areas of enquiry: Aim 1: Engineering and characterization. We will characterize our successfully reengineered, gene-targeted endonucleases in living cells. Using different experimental strategies, we have recently completed the successful selection and redesign of two different endonuclease scaffolds that specifically cleave target sites in (i) the human cystic fibrosis-associated chloride transporter (CFTR) gene and (ii) the human monoamine oxidase (MOAB) gene. Starting with these two enzymes, we will (a) establish the relationship of their in vitro recognition specificity and cleavage activity to their in vivo ability to induce homologous recombination versus nonhomologous end-joining, while (b) simultaneously measuring their toxicity profiles. We will also (c) compare their relative efficiencies of gene conversion when they introduce double strand breaks, versus when engineered nickase versions of the same enzymes introduce single-strand breaks. The constructs mentioned above were produced using two very different methods, each developed for a specific target. In subaim (d), we will continue to improve and combine methods for homing endonuclease redesign. This work involves the iterative application of bioinformatics (to identify new endonuclease scaffolds), computational structure-based design, and directed evolution of targeted cleavage activity. Aim 2: Determination of new endonuclease structures and functions. We will determine the structure of the gp29 endonuclease from bacteriophage 0305f8-36. This protein family was discovered while examining metagenomic sequence data. Its members are shown to display a novel combination of protein domain organization and DNA recognition that is appropriate for several important genomic applications. PUBLIC HEALTH RELEVANCE: The development of highly specific, nontoxic gene targeting proteins that are capable of inducing the site-specific modification of a DNA sequence (either the insertion of novel genes or the disruption or alteration of existing genes) is a critical technology for genome engineering and for corrective gene therapy. Newly discovered endonucleases also provide raw material for the creation of new genome mapping reagents.